Nitrilases, nucleic acids encoding them and methods for making and using them

Abstract

The invention relates to nitrilases and to nucleic acids encoding the nitrilases. In addition methods of designing new nitrilases and method of use thereof are also provided. The nitrilases have increased activity and stability at increased pH and temperature.

What is claimed is: 
     
       1. An isolated, synthetic, or recombinant variant polypeptide having nitrilase activity, wherein the variant polypeptide is at least 95% identical to the full length of SEQ ID NO:210 and has at least one mutation selected from the group consisting of:
 A55G, A55K, I60E, N111S, A190T, A190S, A190H, F191L, F191T, F191M, F191V, M199E, and M199L. 
 
     
     
       2. A method for making an (R)-ethyl 4-cyano-3-hydroxybutyric acid, the method comprising contacting a hydroxyglutaryl nitrile with the polypeptide of  claim 1 , wherein the polypeptide selectively produces an (R)-enantiomer, so as to make (R)-ethyl 4-cyano-3-hydroxybutyric acid. 
     
     
       3. A method for making an (S)-ethyl 4-cyano-3-hydroxybutyric acid, the method comprising contacting a hydroxyglutaryl nitrile with the polypeptide of  claim 1 , wherein the polypeptide selectively produces an (S)-enantiomer, so as to make (S)-ethyl 4-cyano-3-hydroxybutyric acid. 
     
     
       4. A composition comprising the polypeptide of  claim 1 .